A NPAS4-NuA4 complex couples synaptic activity to DNA repair.

Neuronal activity is crucial for adaptive circuit remodelling but poses an inherent risk to the stability of the genome across the long lifespan of postmitotic neurons1-5. Whether neurons have acquired specialized genome protection mechanisms that enable them to withstand decades of potentially damaging stimuli during periods of heightened activity is unknown. Here we identify an activity-dependent DNA repair mechanism in which a new form of the NuA4-TIP60 chromatin modifier assembles in activated neurons around the inducible, neuronal-specific transcription factor NPAS4. We purify this complex from the brain and demonstrate its functions in eliciting activity-dependent changes to neuronal transcriptomes and circuitry. By characterizing the landscape of activity-induced DNA double-strand breaks in the brain, we show that NPAS4-NuA4 binds to recurrently damaged regulatory elements and recruits additional DNA repair machinery to stimulate their repair. Gene regulatory elements bound by NPAS4-NuA4 are partially protected against age-dependent accumulation of somatic mutations. Impaired NPAS4-NuA4 signalling leads to a cascade of cellular defects, including dysregulated activity-dependent transcriptional responses, loss of control over neuronal inhibition and genome instability, which all culminate to reduce organismal lifespan. In addition, mutations in several components of the NuA4 complex are reported to lead to neurodevelopmental and autism spectrum disorders. Together, these findings identify a neuronal-specific complex that couples neuronal activity directly to genome preservation, the disruption of which may contribute to developmental disorders, neurodegeneration and ageing.

Heat Exposure, Heat-Related Symptoms and Coping Strategies among Elderly Residents of Urban Slums and Rural Vilages in West Bengal, India.

The impact of heat stress among the elderly in India-particularly the elderly poor-has received little or no attention. Consequently, their susceptibility to heat-related illnesses is virtually unknown, as are the strategies they use to avoid, or deal with, the heat. This study examined perceptions of comfort, heat-related symptoms, and coping behaviors of 130 elderly residents of Kolkata slums and 180 elderly residents of rural villages south of Kolkata during a 90-day period when the average 24-h heat indexes were between 38.6 °C and 41.8 °C. Elderly participants in this study reported being comfortable under relatively warm conditions-probably explained by acclimatization to the high level of experienced heat stress. The prevalence of most heat-related symptoms was significantly greater among elderly women, who also were more likely to report multiple symptoms and more severe symptoms. Elderly women in the rural villages were exposed to significantly hotter conditions during the day than elderly men, making it likely that gender differences in symptom frequency, number and severity were related to gender differences in heat stress. Elderly men and elderly village residents made use of a greater array of heat-coping behaviors and exhibited fewer heat-related symptoms than elderly women and elderly slum residents. Overall, heat measurements and heat-related symptoms were less likely to be significant predictors of most coping strategies than personal characteristics, building structures and location. This suggests that heat-coping behaviors during hot weather were the result of complex, culturally influenced decisions based on many different considerations besides just heat stress.

Formation of thyroid hormone revealed by a cryo-EM structure of native bovine thyroglobulin.

Thyroid hormones are essential regulators of metabolism, development, and growth. They are formed from pairs of iodinated tyrosine residues within the precursor thyroglobulin (TG), a 660-kDa homodimer of the thyroid gland, by an oxidative coupling reaction. Tyrosine pairs that give rise to thyroid hormones have been assigned within the structure of human TG, but the process of hormone formation is poorly understood. Here we report a ~3.3-Å cryo-EM structure of native bovine TG with nascent thyroid hormone formed at one of the predicted hormonogenic sites. Local structural rearrangements provide insight into mechanisms underlying thyroid hormone formation and stabilization.

Individually experienced heat stress among elderly residents of an urban slum and rural village in India.

The elderly are one of the most vulnerable groups to heat-related illnesses and mortality. In tropical countries like India, where heat waves have increased in frequency and severity, few studies have focused on the level of stress experienced by the elderly. The study presented here included 130 elderly residents of Kolkata slums and 180 elderly residents of rural villages about 75 km south of Kolkata. It used miniature monitoring devices to continuously measure temperature, humidity, and heat index experienced during everyday activities over 24-h study periods, during hot summer months. In the Kolkata slum, construction materials and the urban heat island effect combined to create hotter indoor than outdoor conditions throughout the day, and particularly at night. As a result, elderly slum residents were 4.3 times more likely to experience dangerous heat index levels (≥ 45°C) compared to rural village elderly. In both locations, the median 24-h heat indexes of active elderly were up to 2°C higher than inactive/sedentary elderly (F = 25.479, p < 0.001). Among Kolkata slums residents, there were no significant gender differences in heat exposure during the day or night, but in the rural village, elderly women were 4 times more likely to experience dangerous heat index levels during the hottest times of the day compared to elderly men. Given the decline in thermoregulatory capacity associated with aging and the increasing severity of extreme summer heat in India, these results forecast a growing public health challenge that will require both scientific and government attention.

Circadian hepatocyte clocks keep synchrony in the absence of a master pacemaker in the suprachiasmatic nucleus or other extrahepatic clocks.

It has been assumed that the suprachiasmatic nucleus (SCN) synchronizes peripheral circadian oscillators. However, this has never been convincingly shown, since biochemical time series experiments are not feasible in behaviorally arrhythmic animals. By using long-term bioluminescence recording in freely moving mice, we show that the SCN is indeed required for maintaining synchrony between organs. Surprisingly, however, circadian oscillations persist in the livers of mice devoid of an SCN or oscillators in cells other than hepatocytes. Hence, similar to SCN neurons, hepatocytes can maintain phase coherence in the absence of Zeitgeber signals produced by other organs or environmental cycles.

Using multiple correspondence analysis to identify behaviour patterns associated with overweight and obesity in Vanuatu adults.

OBJECTIVE: The present study evaluates the use of multiple correspondence analysis (MCA), a type of exploratory factor analysis designed to reduce the dimensionality of large categorical data sets, in identifying behaviours associated with measures of overweight/obesity in Vanuatu, a rapidly modernizing Pacific Island country. DESIGN: Starting with seventy-three true/false questions regarding a variety of behaviours, MCA identified twelve most significantly associated with modernization status and transformed the aggregate binary responses of participants to these twelve questions into a linear scale. Using this scale, individuals were separated into three modernization groups (tertiles) among which measures of body fat were compared and OR for overweight/obesity were computed. SETTING: Vanuatu.ParticipantsNi-Vanuatu adults (n 810) aged 20-85 years. RESULTS: Among individuals in the tertile characterized by positive responses to most of or all the twelve modernization questions, weight and measures of body fat and the likelihood that measures of body fat were above the US 75th percentile were significantly greater compared with individuals in the tertiles characterized by mostly or partly negative responses. CONCLUSIONS: The study indicates that MCA can be used to identify individuals or groups at risk for overweight/obesity, based on answers to simply-put questions. MCA therefore may be useful in areas where obtaining detailed information about modernization status is constrained by time, money or manpower.

Coming to grips with economic development: Variation in adult hand grip strength during health transition in Vanuatu.

OBJECTIVES: To determine whether (1) maximal handgrip strength (HGS) is associated with inter-island level of economic development in Vanuatu, (2) how associations between island of residence and HGS are mediated by age, sex, body size/composition, and individual sociodeomographic variation, and (3) whether HGS is predictive of hypertension. MATERIAL AND METHODS: HGS was collected from 833 adult (aged 18 and older) men and women on five islands representing a continuum of economic development in Vanuatu. HGS was measured using a handheld dynamometer. Participants were administered in an extensive sociobehavioral questionnaire and were also assessed for height, weight, percent body fat, forearm skinfold thickness, forearm circumference, and blood pressure. RESULTS: HGS was significantly greater in men than in women regardless of island of residence. HGS was also significantly positively associated with inter-island level of economic development. Grip strength-to-weight ratio was not different across islands except in older individuals, where age-related decline occurred primarily on islands with greater economic development. HGS significantly declined with age in both men and women. CONCLUSION: HGS is positively associated with modernization in Vanuatu, but the relationship between HGS and modernization is largely due to an association of both variables with increased body size on more modernized islands. Further research on the role of individual variation in diet and physical activity are necessary to clarify the relationship between HGS and modernization.

Macromolecular Assemblies of the Mammalian Circadian Clock.

The mammalian circadian clock is built on a feedback loop in which PER and CRY proteins repress their own transcription. We found that in mouse liver nuclei all three PERs, both CRYs, and Casein Kinase-1δ (CK1δ) are present together in an ∼1.9-MDa repressor assembly that quantitatively incorporates its CLOCK-BMAL1 transcription factor target. Prior to incorporation, CLOCK-BMAL1 exists in an ∼750-kDa complex. Single-particle electron microscopy (EM) revealed nuclear PER complexes purified from mouse liver to be quasi-spherical ∼40-nm structures. In the cytoplasm, PERs, CRYs, and CK1δ were distributed into several complexes of ∼0.9-1.1 MDa that appear to constitute an assembly pathway regulated by GAPVD1, a cytoplasmic trafficking factor. Single-particle EM of two purified cytoplasmic PER complexes revealed ∼20-nm and ∼25-nm structures, respectively, characterized by flexibly tethered globular domains. Our results define the macromolecular assemblies comprising the circadian feedback loop and provide an initial structural view of endogenous eukaryotic clock machinery.

Rolling Tobacco in Banana Leaves, Newspaper, or Copybook Paper Associated With Significant Reduction in Lung Function in Vanuatu.

In addition to the widespread availability of packaged cigarettes, the inhabitants of island nations of the Southwest Pacific frequently smoke commercially available loose tobacco using manufactured rolling papers, as well as locally grown tobacco rolled in manufactured rolling paper or wrapped in leaves, copybook paper, and newspaper. In this study, Vanuatu men who smoked local tobacco rolled in leaves, copybook paper, or newspaper showed significantly lower forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), and FEV1/FVC ratios than men who smoked packaged cigarettes, store-bought tobacco rolled in manufactured rolling paper, or who smoked locally grown tobacco rolled in manufactured rolling papers. The addition of toxins from these unusual tobacco-wrapping media produces lung function deficits similar to the pattern noted among tobacco smokers who also inhale smoke from burning biomass. Thus, public health initiatives should consider including strategies addressing the use of wrapping media among smokers in South Pacific island societies.

Secular change in adult stature associated with modernization in Vanuatu.

OBJECTIVE: To determine whether: (1) there is a secular increase in adult stature in Vanuatu, and (2) whether adult stature is positively associated with modernization in Vanuatu. METHODS: This study reports on stature measurements collected on 650 adult (age > 17 years) men and women from four islands of varying economic development in Vanuatu. Measurements were collected as part of the Vanuatu Health Transitions Research Project in 2007 and 2011. RESULTS: Stature increased significantly in adults born between the 1940s and 1960s in Vanuatu, before leveling off in those born between the 1970s and 1990s. Adults are significantly taller on Efate, the most modernized island in the study sample, than on the less economically developed islands. CONCLUSIONS: Modernization is likely associated with improvements in child growth in Vanuatu, as assessed by gains in adult stature.

Ownership of consumer electronics is associated with measures of adiposity during health transition in Vanuatu.

OBJECTIVE: The Republic of Vanuatu, like many developing nations, is undergoing a rapid health transition. Our previous study identified several behavioral risk factors for the rising prevalence of obesity. Unexpectedly, daily time spent using television and radio was revealed as a protective factor for obesity in 2007. In this study, we sought to explore associations between ownership of consumer electronics (CE) and measures of adiposity in Vanuatu in 2011. METHODS: We surveyed 873 adults from five islands varying in level of economic development. Height, weight, and waist circumferences; triceps, subscapular, and suprailiac skinfolds; and percent body fat by bioelectrical impedance were measured. Ownership of eight types of CE, diet through 24-h dietary recall and leisure-time activity patterns were assessed using a questionnaire. RESULTS: Participants from more developed islands owned more types of CE, and revealed higher measures of adiposity on average as well as higher prevalence of obesity/central obesity. When controlling for demographic factors, and dietary and activity patterns, increased measures of adiposity and risk for obesity/central obesity were associated with ownership of cellphones, music players, televisions, video players, microwaves, and/or refrigerators. Positive correlations between CE ownership and measures of adiposity were mainly observed among men on the two most developed islands. CONCLUSIONS: The results of this study indicate a possible role of CE use in the rising prevalence of obesity and the shift to a sedentary lifestyle in Vanuatu and many other modernizing regions, where prevention efforts including education on healthy use of CE are imperative.

Larger FVC and FEV1 among Tibetans compared to Han born and raised at high altitude.

OBJECTIVES: This study compares forced vital capacity (FVC) and Forced Expiratory Volume at 1 Second (FEV1 ) of Tibetans with those of Han who were born and raised at high altitude. MATERIALS AND METHODS: FVC and FEV1 tests were conducted among 1,063 children and adolescents between the ages of 6 and 20 years, and 184 adults between the ages of 21 and 39 years who had lived their entire lives at 3200 m, 3800 m and 4300 m in Qinghai Provence, Peoples Republic of China. RESULTS: Even though FVC and FEV1 values of Han born and raised at high altitude are generally lower than those of Tibetans through age 15 in girls and age 16 in boys, differences are largely explained by variation in stature (height-squared) and chest circumference. Among older adolescents and adults, the FVC and FEV1 values of Tibetans are significantly larger than those of Han born and raised at high altitude; and are much larger than would be predicted, based on stature and chest circumference. DISCUSSION: These results indicate that the large FVC and FEV1 values of Tibetan adults develop primarily from an accelerated pattern of lung growth that begins during mid-to-late adolescence and possibly extends into young adulthood. This developmental pattern is not only distinct from that of Han born and raised at high altitude, but also from those of Andean Quechua and Aymara. The pace of lung function growth may therefore represent another feature distinguishing the Tibetan from the Andean pattern of adaptation to high altitude hypoxia. Because of this, a search for features in the Tibetan genome related to this lung function growth phenotype might be productive and important.

Stunting and the Prediction of Lung Volumes Among Tibetan Children and Adolescents at High Altitude.

This study examines the extent to which stunting (height-for-age Z-scores ≤ -2) compromises the use of low altitude prediction equations to gauge the general increase in lung volumes during growth among high altitude populations. The forced vital capacity (FVC) and forced expiratory volume (FEV1) of 208 stunted and 365 non-stunted high-altitude Tibetan children and adolescents between the ages of 6 and 20 years are predicted using the Third National Health and Nutrition Examination Survey (NHANESIII) and the Global Lung Function Initiative (GLF) equations, and compared to observed lung volumes. Stunted Tibetan children show smaller positive deviations from both NHANESIII and GLF prediction equations at most ages than non-stunted children. Deviations from predictions do not correspond to differences in body proportions (sitting heights and chest circumferences relative to stature) between stunted and non-stunted children; but appear compatible with the effects of retarded growth and lung maturation that are likely to exist among stunted children. These results indicate that, before low altitude standards can be used to evaluate the effects of hypoxia, or before high altitude populations can be compared to any other group, it is necessary to assess the relative proportion of stunted children in the samples. If the proportion of stunted children in a high altitude population differs significantly from the proportion in the comparison group, lung function comparisons are unlikely to yield an accurate assessment of the hypoxia effect. The best solution to this problem is to (1) use stature and lung function standards based on the same low altitude population; and (2) assess the hypoxic effect by comparing observed and predicted values among high altitude children whose statures are most like those of children on whom the low altitude spirometric standard is based-preferably high altitude children with HAZ-scores ≥ -1.

Histone monoubiquitination by Clock-Bmal1 complex marks Per1 and Per2 genes for circadian feedback.

Circadian rhythms in mammals are driven by a feedback loop in which the transcription factor Clock-Bmal1 activates expression of Per and Cry proteins, which together form a large nuclear complex (Per complex) that represses Clock-Bmal1 activity. We found that mouse Clock-Bmal1 recruits the Ddb1-Cullin-4 ubiquitin ligase to Per (Per1 and Per2), Cry (Cry1 and Cry2) and other circadian target genes. Histone H2B monoubiquitination at Per genes was rhythmic and depended on Bmal1, Ddb1 and Cullin-4a. Depletion of Ddb1-Cullin-4a or an independent decrease in H2B monoubiquitination caused defective circadian feedback and decreased the association of the Per complex with DNA-bound Clock-Bmal1. Clock-Bmal1 thus covalently marks Per genes for subsequent recruitment of the Per complex. Our results reveal a chromatin-mediated signal from the positive to the negative limb of the clock that provides a licensing mechanism for circadian feedback.

Single-cell analysis of circadian dynamics in tissue explants.

Tracking molecular dynamics in single cells in vivo is instrumental to understanding how cells act and interact in tissues. Current tissue imaging approaches focus on short-term observation and typically nonendogenous or implanted samples. Here we develop an experimental and computational setup that allows for single-cell tracking of a transcriptional reporter over a period of >1 wk in the context of an intact tissue. We focus on the peripheral circadian clock as a model system and measure the circadian signaling of hundreds of cells from two tissues. The circadian clock is an autonomous oscillator whose behavior is well described in isolated cells, but in situ analysis of circadian signaling in single cells of peripheral tissues is as-yet uncharacterized. Our approach allowed us to investigate the oscillatory properties of individual clocks, determine how these properties are maintained among different cells, and assess how they compare to the population rhythm. These experiments, using a wide-field microscope, a previously generated reporter mouse, and custom software to track cells over days, suggest how many signaling pathways might be quantitatively characterized in explant models.

Impact of modernization on adult body composition on five islands of varying economic development in Vanuatu.

OBJECTIVE: The Republic of Vanuatu, similar to other South Pacific island nations, is undergoing a rapid health transition as a consequence of modernization. The pace of modernization is uneven across Vanuatu's 63 inhabited islands, resulting in differential impacts on overall body composition and prevalence of obesity among islands, and between men and women. In this study, we investigated (1) how modernization impacts body composition between adult male and female Melanesians living on four islands of varying economic development in Vanuatu, and (2) how body composition differs between adult Melanesians and Polynesians living on rural islands in Vanuatu. METHODS: Anthropometric measurements were taken on adult male and female Melanesians aged 18 years and older (n = 839) on the islands of Ambae (rural), Aneityum (rural with tourism), Nguna (rural with urban access), and Efate (urban) in Vanuatu, in addition to Polynesian adults on Futuna (rural). RESULTS: Mean measurements of body mass and fatness, and prevalence of obesity, were greatest on the most modernized islands in our sample, particularly among women. Additionally, differences between men and women became more pronounced on islands that were more modernized. Rural Polynesians on Futuna exhibited greater body mass, adiposity, and prevalence of obesity than rural Melanesians on Ambae. CONCLUSIONS: We conclude that Vanuatu is undergoing an uneven and rapid health transition resulting in increased prevalence of obesity, and that women are at greatest risk for developing obesity-related chronic diseases in urbanized areas in Vanuatu.

Purification and analysis of PERIOD protein complexes of the mammalian circadian clock.

In mammals, circadian rhythms are generated at least in part by a cell-autonomous transcriptional feedback loop in which the three PERIOD (PER) and two CRYPTOCHROME (CRY) proteins inhibit the activity of the dimeric transcription factor CLOCK-BMAL1, thereby repressing their own expression. Upon nuclear entry, the PER and CRY proteins form a large protein complex (PER complex) that carries out circadian negative feedback by means of at least two basic functions: (1) it brings together multiple effector proteins that repress transcription and (2) it delivers these repressive effectors directly to CLOCK-BMAL1 bound to E-box sequences of circadian target genes. At present, the composition, mechanisms of action, and dynamics of PER complexes in circadian clock negative feedback are incompletely understood. Here, we describe several experimental approaches to the study of PER complexes obtained from mammalian tissues. We focus on the isolation of nuclei from mouse tissues, the extraction of PER complexes from the isolated nuclei, characterization of native PER complexes by gel filtration and blue native polyacrylamide gel electrophoresis, preparative immunoaffinity purification of PER complexes for mass spectrometric identification of constituent proteins, and chromatin immunoprecipitation to monitor the recruitment of PER complex proteins to CLOCK-BMAL1 at E-box sites of clock-regulated genes.

Specificity in circadian clock feedback from targeted reconstitution of the NuRD corepressor.

Mammalian circadian rhythms are generated by a negative feedback loop in which PERIOD (PER) proteins accumulate, form a large nuclear complex (PER complex), and bind the transcription factor CLOCK-BMAL1, repressing their own expression. We found that mouse PER complexes include the Mi-2/nucleosome remodelling and deacetylase (NuRD) transcriptional corepressor. Unexpectedly, two NuRD subunits, CHD4 and MTA2, constitutively associate with CLOCK-BMAL1, with CHD4 functioning to promote CLOCK-BMAL1 transcriptional activity. At the onset of negative feedback, the PER complex delivers the remaining complementary NuRD subunits to DNA-bound CLOCK-BMAL1, thereby reconstituting a NuRD corepressor that is important for circadian transcriptional feedback and clock function. The PER complex thus acquires full repressor activity only upon successful targeting of CLOCK-BMAL1. Our results show how specificity is generated in the clock despite its dependence on generic transcriptional regulators and reveal the existence of active communication between the positive and negative limbs of the circadian feedback loop.

Adult lipids associated with early life growth in traditional Melanesian societies undergoing rapid modernization: a longitudinal study of the mid-20th century.

Both poor fetal development and accelerated post-natal growth have been linked to adult dyslipidemias in many studies conducted in developed societies. It is not known, however, whether these relationships only characterize populations with typical Western diets or if they also may develop in groups at the early stages of a dietary transition. Our longitudinal study of traditional rural populations in the Southwest Pacific during a period of extremely rapid modernization in diet and life-styles shows a nascent association between child growth retardation, subsequent growth acceleration, and adult lipid values in spite of a continuing prevalence of very low lipid levels. However, our results do not entirely conform to results from populations with "modern" diets. Outcome (i.e., young adult) cholesterol and triglyceride levels are more consistently related to initial measures of body fat and growth in body fat measures than with stature, while outcome apo A-1 is more consistently related to initial stature or stature growth than to measures of body fat. We suggest this may reflect a pattern characteristic of the initial stages of "modernization" associated with dietary change, with stronger and more pervasive relationships emerging only later as populations complete the dietary transition.

Temporal orchestration of repressive chromatin modifiers by circadian clock Period complexes.

The mammalian circadian clock is built on a molecular feedback loop in which the Period (PER) proteins, acting in a large, poorly understood complex, repress Clock-Bmal1, the transcription factor driving their expression. We found that mouse PER complexes include the histone methyltransferase HP1γ-Suv39h. PER proteins recruited HP1γ-Suv39h to the Per1 and Per2 promoters, and HP1γ-Suv39h proved important for circadian di- and trimethylation of histone H3 Lys9 (H3K9) at the Per1 promoter, feedback repression and clock function. HP1γ-Suv39h was recruited to the Per1 and Per2 promoters ~4 h after recruitment of HDAC1, a PER-associated protein previously implicated in clock function and H3K9 deacetylation at the Per1 promoter. PER complexes containing HDAC1 or HP1γ-Suv39h appeared to be physically separable. Circadian clock negative feedback by the PER complex thus involves dynamic, ordered recruitment of repressive chromatin modifiers to DNA-bound Clock-Bmal1.